1. Field of the Invention
The present invention relates to a method for mass production of paclitaxel, a secondary metabolite by plant cell culture.
2. Description of the Prior Art
Paclitaxel is one of taxane compounds which are isolated from the bark of Taxus brevifolia, and it is effective for the treatment of cancer, such as leukemia. It is reported that paclitaxel is capable of curing approximately 30%, 50% and 20% of ovarian, breast and lung cancers, respectively, by inhibiting depolymerization of microtubules. Generally, paclitaxel can be produced by a total chemical synthesis, by a semi-synthesis employing precursors such as baccatin, direct extraction of paclitaxel from Taxus genus plants or by culturing cells that produce paclitaxel. Among these methods, plant cell culture-based process for paclitaxel production has the following advantages. First, paclitaxel can be produced in a continuous manner regardless of a fluctuation in the supply of yew plants due to damages by blight, and harmful insects or by natural disasters. Secondly, cell cultures can be propagated in large bioreactors, from which paclitaxel can be massively produced by manipulating culture conditions. Thirdly, cell cultures produce a simpler spectrum of compounds compared to other methods, considerably simplifying separation and purification. Fourthly, a cell culture process can adapt quickly to rapid changes in demand better than the other methods. And fifthly, a cell culture process can produce paclitaxel as well as taxane precursors such as baccatin that can be converted to paclitaxel.
Methods for producing paclitaxel by utilizing plant cell culture have been described in the art:
U.S. Pat. No. 5,019,504 discloses a method for producing paclitaxel and its derivatives utilizing cultured cells of Taxus brevifolia. The yield of paclitaxel described therein, however, is 1xcx9c3 mg/L with the doubling time for the biomass is 7xcx9c12 days which is insufficient for industrial application. Moreover, the production of paclitaxel by the plant culture is unstable and even when a primary cell having high production level is obtained by selection, it is difficult to keep its content by subculturing (E. R. M. Wickremesine et. al., World Congress on Cell and Tissue Culture (1992)).
WO 93/17121 offers a method for paclitaxel production by cell culture of Taxus genus plant while changing composition of the medium, growth rate, and production rate, etc. In case of Taxus chinensis, 24.1 mg/L of paclitaxel can be obtained in 18 days of culture and biomass doubles every 2.5 days.
All of these patents describe methods for mass production of paclitaxel by controlling the cell cultivation temperature to 25xc2x0 C.; there are no teaching in said patents, nor do they anticipate that the changes in the growth rate or production rate when the cell cultivation temperature is varied.
The object of the present invention is to provide a convenient and efficient method for mass production of paclitaxel.
Another object of the present invention is to provide a convenient method for the production of paclitaxel with high yield by regulating the temperature during the plant cell culture.
Another object of the present invention is to provide a more productive method of producing paclitaxel by inoculating a strain that produces paclitaxel at a high concentration.